Magnetic resonance imaging (MRI) is a technique widely used in the medical field, in particular for diagnostic purposes. MRI is based mainly on the observation of the magnetization of the spins of the nuclei of hydrogen atoms in water molecules. It consists in measuring the relaxation by two modes (T1 and T2) of the water molecules with respect to a distribution at equilibrium, in order to form an image. The relaxation occurs after a state in which all the spins have been completely aligned under the effect of an external magnetic field by use of a radiofrequency pulse. MRI makes it possible to distinguish between the regions of high concentration and the regions of low concentration of a contrast agent. In the biological field, the image obtained reveals the tissues rich in contrast agent, essentially those which are hydrophilic in nature.
First-generation contrast agents are “passive” agents which are distributed through the sample subjected to MRI, essentially as a function of lipophilicity gradients and nonspecific bonds.
Studies have been carried out to define active agents, that is to say agents capable of revealing the presence of a chemical or biochemical activity, pH conditions or a concentration of a specific ion. These agents are composed of a strongly paramagnetic ion complexed by a ligand, the ion generally being Gd(III). The contrast agents comprising such a strongly paramagnetic ion consequently constitute a permanent molecular magnet which always has a greater or lesser effect on the relaxation of the surrounding water molecules. The means for rendering these complexes of permanent molecular magnet type sensitive to specific chemical compounds of their environment was based essentially on the modification of the accessibility of the immediate environment of the paramagnetic metal for water molecules. Such labels, for which the relaxation by an enzymatic activity is improved in that they act as catalysts for the rapid exchange of water molecules are described in various documents.
U.S. Pat. No. 5,707,605 discloses an MRI agent which is a complex of a paramagnetic metal ion and of a ligand, said ligand comprising a chelating group and a substituent which is covalently bonded to the chelating group. In the absence of target substance, the substituent is combined with or bonded to the metal ion of the complex and it occupies or blocks at least one coordination site of the metal ion. All the coordination sites of the metal ion are then saturated by the chelating group and the substituent. In the presence of the target substance, the substituent reacts with said substance and ceases to block or to occupy at least one coordination site of the metal ion, so that a rapid exchange of water can take place on the site, which improves the MR image. A derivative of 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) in which one of the acetate substituents is replaced by a hydroxyethyl β-galactose substituent is an example of such a complex. In this complex, the end group sensitive to the enzyme has only a slight protective effect on the metal center with regard to coordination by water: the mean number of water molecules which are coordinated at any moment to the 9th coordination site of the gadolinium is 0.65 instead of 1.02 for the “cleaved” form of a similar compound comprising a ligand capable of occupying only 8 coordination sites (Cf. Meade, T. J. et al., “In vivo visualization of gene expression using magnetic resonance imaging. Nature Biotechnology, 18, 321-325 (2000)”). This low additional access of the water molecules during the bioactivation of the contrast agent does not satisfactorily modify the relaxation.
In addition, the Applicant Company has discovered another disadvantage related to this contrast agent, namely that the latter does not react with a sufficient speed with the target substance, namely the enzyme β-galactosidase. This is because the Applicant has carried out experiments which have shown that coinjection of this same contrast agent with the enzyme into a living test organism, namely zebra fish embryos, does not result in any modification in the contrast, neither after 24 hours nor after 48 hours. This result was obtained in comparison with an identical experiment in which the coinjected agent was a commercial chromogenic agent which gave rise to an intense coloring of the embryo related to its enzymatic conversion.
WO 99/21592 discloses the application of the technique of U.S. Pat. No. 5,707,605 to a target substance of the therapeutic agent type. The improvement in the degree of contrast which can be obtained using the labels of the type of those disclosed in the abovementioned documents is, however, limited.
Complexes formed by a transition metal ion and by a ligand which can exist in a high spin form and in a low spin form, depending on the nature of the ligand, are known. In the high spin state, virtually all the electrons are unpaired electrons; there is no internal compensation of their spins, which produces a substantial increase in the magnetic moment. In the low spin state, the complexes have a weak, indeed even zero, magnetic moment, depending on the number of unpaired electrons (generally 0 or 1). An example of Fe(II) compound complexed by a hexadentate macrocyclic ligand of low spin is given by Wieghardt et al. (Inorg. Chem., 1986, 25, 4877) and an example of an Fe(II) compound complexed by a similar pentadentate macrocyclic ligand of high spin is given by Spiccia et al. (Inorg. Chim. Acta, 1998, 279, 192).